CA2124027A1 - Dna originating in a non-a non-b hepatitis virus gene and constituting polypeptide - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE

The present invention provides DNAs originating in non-A non-B hepatitis virus genome gene and polypeptides constituting said virus, which enable the diagnosis of hepatitis induced by said virus. An RNA is extracted and purified from the plasma of a patient with non-A non-B
hepatitis. By using this RNA as a template, a single strand DNA is prepared. Then, the DNA is amplified by the PCR method with the use of appropriately selected primers.
The partial DNA sequence of the above-mentioned virus is identified by analyzing the structure and the constituting polypeptide of this virus is deduced. Thus, the antigen site is confirmed.

Description

212 ~1 0 ;;~ r~

SPECIFICATION

DNA ORIGINATING IN NON-A NON-B HEPATITIS ~
VIRUS GENE AND CONSTITUTING POLYPEPTIDE ~-Background of the Invention Field of the Invention The present invention relates to polypeptides which relate to non-A non-B hepatitis virus and DNAs encoding for the same. Further, the present invention relates to reagents for detecting anti non-A non-B ;-hepatitis virus antibody and non-A non-B hepatitis virus gene. ~ :
Description of the Related Art Non-A non-B hepatitis is thought to amount to ~ -about 95 % of posttransfusive hepatitis at the present stage. Thus it has been urgently required to find out patients infected with non-A non-B hepatitis virus in -order to exclude their blood from the blood to be transfused and to develop a vaccine for preventing this disease. Recently, Houghton et al. have reported the isolation of a gene of this virus [see European Patent Publication-A No. 318216 (published on May 31, 1989]] and marketed a reagent for detecting anti non-A
non-B hepatitis virus antibody with the use of this 212'~.021~

recombinant viral antigen. Although it is expected that posttransfusive non-A non-B hepatitis would be prevented by finding blood wherein anti non-A non-B
hepatitis virus antibody is present with this antibody detection reagent and excluding such blood from the blood to be transfused. However, this hepatitis occurs at an incidence of 5 to 7 % even after excluding the blood of anti non-A non-B hepatitis virus antibody positive donors. Thus, this disease has not been exterminated yet.
Indeed, one of the possible reasons therefor resides in the fact that the relation between the infection with this virus and the production of the antibody against this virus still remains unelucidated. However, another reason therefor resides in the finding that the nucleic acid sequence of the genome of this virus seemingly has a considerably high heterogeneity. This finding is based on the results of a number of experiments wherein c-DNA of this virus genome was cloned by the PCR method or the prlmer extension method based on the nucleic acid sequence reported by Houghton et al. [see Kubo et al., Nucleic Acid Research, 17, 10367 - 10372 (1989); Kato et al., Proc. Japan Acad., ~, 219 - 223 (1989); Maeno et al., Nucleic Acid Research, 18, 212~02~ ~

2685 - 2~89 (1990); Kato et al., Proc. Natl. Acad.
Sci. USA, ~7, 9524 - 9528 (1990)]. Accordingly. it may not be concluded that the diagnosis of non-A non-B
hepatitis and the prevention against the onset of non-A non-B hepatitis caused by transfusion have been brought to completion and it has been thus required to ~ :
develop a novel diagnostic method therefor including gene diagnosis.
Independently of Houghton et al. but at the same time, Terukatsu Arima, who is one of the present inventors, cloned one of epitopes in the core region of HCV (hepatitis C virus) by the immunoscreening method with the use of the serum of a human patient with non-A non-B hepatitis as an RNA source and an ~-~
antibody source [see Arima et al., Gastroenterologia Japanica. ~, 218 - 222 (1990)].

Disclosure of the Ivention Summary of the Invention The present invention aims at providing a novel antigen polypeptide against non-A non-B hepatitis virus, DNA encoding the polypeptide and a diagnostic reagent for non-A non-B hepatitis with the use of the antigen polypeptide or the DNA.
Under the above-mentioned circumstances, the :~ - - .. . .. . . . .

212~02'~

present inventors have started to study non-A non-B
hepatitis virus. As a result of their extensive studies by using the plasmas of donors in Japan with a high s-GPT level, they have successfully isolated DNAs originating in novel non-A non-B hepatitis virus differing from the virus reported hitherto and confirmed the structures thereof. They have further Pound that non-A non-B hepatitis can be diagnosed by using these DNAs and polypeptides encoded thereby, thus completing the present invention.
Accordingly, the present invention relates to a non-A non-B hepatitis virus-constituting polypeptide represented by SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID
NO: 3, and a polypeptide comprising a part of the amino acid sequence described in SEQ ID NO: 1, SEQ ID
NO: 2 or SEQ ID NO: 3. That is, the present invention provides a polypeptide comprising at least 6 consecutive amino acids in the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID
NO: 3.
Further, the present invention relates to DNA
(full length DNA) encoding the non-A non-B hepatitis virus-constituting polypeptide of the present invention, and DNA comprising a part of the base sequence of the above-mentioned full length DNA. That -. :.

2~2~02~
. - .:

is, the present invention provides DNA comprising at least 10 consecutive bases in the base sequence represented by SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID
NO 6.
Furthermore, the present invention relates to a reagent for detecting anti non-A non-B hepatitis virus antibody by using a part of the above-mentioned non-A
non-B hepatitis virus constituting polypeptide and `
confirming the presence of the anti non-A non-B
hepatitis virus antibody in a sample by an immunological means, a reagent for detecting non-A
non-B hepatitis virus gene by using a part of the above-mentioned full length DNA as a primer to amplifY
DNA originating in non-A non-B hepatitis virus and effecting the detection of the DNA, and a reagent for detecting non-A non-B hepatitis virus gene by using a part of the above-mentioned full length DNA as probe to effect hybridization with a DNA originating in non-A non-B hepatitis virus. That is, the present invention provides an immunochemical reagent for detecting anti non-A non-B hepatitis virus antibody in a biological sample which contains the polypeptide(s) of the present invention, and a reagent for detecting non-A non-B hepatitis virus gene which contains the DNA of the present invention.

2l2~a2~

In addition, the present invention relates to a polyclonal antibody or a monoclonal antibody against the above-mentioned non-A non-B hepatitis virus constituting polypeptide as an antigen, a reagent for detecting non-A non-B hepatitis virus antigen by using the above-mentioned antibody and confirming the presence of the non-A non-B hepatitis virus antigen in a sample by an immunochemical means, and a non-A non-B
hepatitis vaccine produced by using the above-mentioned antigen polypeptide. That is, the present invention provides a polyclonal antibody against the polypeptide of the present invention as an antigen, a monoclonal antibody against the polypeptide of the present invention as an antigen, and a non-A non-B
hepatitis virus vaccine produced by using the polypeptide of the present invention.
Further scope and applicability of the present invention will become apparent from the detailed description given hereinafter. However, it should be ;
understood that the detailed description and specific examples, while indicating preferred embodiments of the invention, are ~iven by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed ' ' ''..~

2 1 2 ~

description. ;~
Detailed Description of the Invention Now, the present invention will be described in greater detail.
(1) Preparation of RNA of non-A non-B hepatitis virus As a material from which an RNA of non-A non-B
hepatitis virus is extracted, the plasma of a donor with a high s-GTP level is usable. A viral fraction is collected by adding polyethylene glycol to the plasma and centrifuging it in accordance with a known method. From this viral fraction, the RNA may be extracted with, for example, a solution containing ~-guanidine thiocyanate, a surfactant, a chelating agent and a reducing agent. That is, the purified RNA can be prepared by extracting the RNA from the viral fraction with the solution described above, and then with phenol, fractionating the extract with an organic solvent [see Chamezynski et al., Anal. Biochem., 16~, -156 (1987)], and subjecting the fraction containing the RNA to density gradient ultracentrifugation.
(2) Preparation and cloning of double stranded DNA
By using the obtained RNA as a template, a double-stranded DNA can be prepared by the conventional methods such as the method for synthesizing a cDNA with the use of, for example, a ~ ' ~2~)2 ~

random primer, a reverse transcriptase and DNA
polymerase [see Gubler, U., et al., Gene, 2~. 263 (1983)].
In accordance with the conventional method, the double-stranded DNA thus obtained is integrated into a bacteriophage such as Azap and Agtll, Escherichia coli as a feeder bacterium is infected with the bacteriophage having the double-stranded DNA
integrated thereinto, and then the Escherichia coli, i.e., the transformant thus obtained, is incubated~.
After screening, the target clone can be obtained. As an example of the method for integrating the double-stranded DNA into the phage vector, a method of Hyunh, T.V., et al. described in DNA Cloning, a practical approach, 1, 49 (1985) may be cited.
The screening of the target clone may be effected in accordance with a known method, for example, as follows. When the double-stranded DNA is integrated into a phage vector Agtll, E. coli Y1090 is infected with this vector and polypeptides contained in the plaques thus formed are transfered onto a nitrocellulose membrane. Next, this transfered membrane is subjected to a screening by an immunochemical means with the use of the serum of a patient with non-A non-B viral hepatitis as an ,'' ' ' ' ' ' '.' . '' '' . ` ' ':' '' '' ' ., . ' ' ,, . "'. '. , ', ~' ' . ' ' ' " . ' :

r~
21~2i~ , antibody source.
However, it is difficult to analyze full sequence of the DNAs originating in non-A non-B hepatitis virus by this method. Therefore, the following method may be applied thereto.
First, an RNA is prepared from the fresh plasma of a patient who has been identified as having infection with non-A non-B hepatitis virus by the above-mentioned method. Then, DNAs are amplified by the conventional PCR method with the use of a single-strand DNA constructed by using the above-mentioned -RNA as a template and an appropriate primer of a known structure, followed by subcloning. Thus, a DNA
originating in non-A non-B hepatitis virus can be obtained. As the primer, an oligonucleotide, which is synthesized by appropriately selecting a well- ~ -maintained part on the basis of the known non-A non-B
hepatitis virus DNA sequence, is usable. Usually, a ~ragment comprising 10 to 30 bases, which are derived -from the non-A non-B hepatitis virus DNA, is used as the primer. The primers employed in the present invention are oligonucleotides synthesized based on the base sequences of HC-J8 [see Okamoto, H., et al., Virology, 188, 331 - 341 (1992)], H'~V-J [see Kato, N., et al., Proc. Natl. Acad. Sci. USA, ~7, 9524 - 9528 g 2 1 2 ~

(1990)] and HC-J6 [see Okamoto, H., et al., J. Gen.
Virol., 7~, 2697 - 2704 (1991)] which have been already reported. Further, the partial base sequences of non-A non-B hepatitis virus was determined by experiments with the use of the primers described above and oligonucleotides each having a base sequence of the appropriately selected site in the partial base sequences of non-A non-B hepatitis virus thus ~ -~
determined, were synthesized and employed as primers.
The structure of a base sequence is identified by the Maxam-Gilbert method [see Maxam, A. M., and Gilbert, W., Proc. Natl. Acad. Sci. USA, 74, 560 (1977)] or the dideoxy method [see Sanger, F., Proc.
Natl. Acad. Sci. USA, 74, ~463 (1977)].

(3) Detection of anti non-A non-B hepatitis virus antibody and reagent therefor By using the non-A non-B hepatitis virus-constituting polypeptide of the present invention or a part of the same as an epitope, the existence of the anti non-A non-B hepatitis virus antibody in a biological sample, for example, the serum of a patlent can be examined by an immunochemical means, which enables the diagnosis of the infection with this -virus. The term "epitope" means an antigen determinant of a polypeptide. It is well known that - 10 - , ~2~ ~2 ~

an epitope generally consists of at least 5 amino acids and a polypeptide consisting of 6 amino acid binds to an antibody [see European Patent Publication-A No. 1388~5 (published on May 2, 1985)]. As the epitope to be used herein, polypeptides comprising at least 3 to 5, preferably at least 8 to 10 and still more preferably at least 11 to 20 consecutive amino acids, which are based on the amino acid sequences described in SEQ ID N0: 1, SEQ ID N0: 2 and SEQ ID N0:
3, may be appropriately selected. Needless to say, even a polypeptide comprising about 20 or more amino acids may be used therefor.
As examples of the immunochemical assay methods, known ones such as the ELISA method, the Western blotting method and the agglutination method may be cited. From the viewpoints of convenience and utility, the ELISA method is preferred. The ELISA
method may be effected in, for example, the following manner. A specimen is added to wells each comprising a solid phase having one or more epitopes adsorbed thereonto and reacted. After washing the wells, an enzyme-labeled anti-human IgG monoclonal antibody is added to the wells to react. After washing the wells, the amount of the binding enzyme is determined. Also cysteine may be added to the N-terminus of the epitope . ~ . . . ........... , - ~. . . ,. , .:, - '' :

.. .- . . ........... ~ -.. - .. : .: - .:

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to thereby facilitate the adsorption of the epitope onto the solid phase.
As the substance for labeling the anti-human IgG
antibody, any substances such as fluorescent substances, radioactive substances, bio- or chemiluminescent substances, electrochemical luminescent substances, pigments, enzymes and metals . -are usable, so long as it can be assayed. It is preferable to use enzymes and electrochemical -luminescent substances therefor.
As the solid support for the epitope, microtiter plates, plastic beads, erythrocytes, gelatin particles, latex particles and magnetic particles may be cited and everyone are usable.
To select the epitope, it is preferable that site suitable as the epitope is confirmed by using the sera of a number of patients with non-A non-B hepatitis on the basis of the ELISA method.
Now, a particular example of the assay reagent of the present invention will be described. The assay reagent of the present invention is in the form of kit which contains, as the essential component(s), one or more polypeptides each comprising the full length of a non-A non-B hepatitis virus-constituting polypeptide or a part thereof as its epitope, and at ;~

..

~ ~ 2 ~ 0 2 1 least one additional component(s) arbitrarily selected from among standard sera of patients with non-A non-B
hepatitis virus (antibody), anti-human IgG antibody, enzymes and enzyme substrates. When this kit further contains a solid phase, this solid phase may be provided in a state of being coated with the polypeptide comprising the epitope. When it contains anti-human IgG antibody and an enzyme, these substances ~ay be provided in a conjugated state.
These kits also fall within the scope of the embodiment of the assay reagent of the present invention. Further, the kit may contain appropriate an antigen diluent, a reaction diluent, a solvent for a substrate or a reaction stopper solution for the convenience in the embodiment of the assay. These embodiments should not be considered to limit the present invention.
,he specimen to be assayed is a so-called biological sample such as serum, plasma, spinal fluid, lymph, saliva and cells, though the specimen is not restricted thereto.

(4) Preparation on of anti non-A non-B hepatitis virus polypeptide antibody An antibody can be prepared by using, as the antigen, a polypeptide or a protein comprising the ' . . ',.. '., , . ' .:, . ~,.. ; , ..

~l2llQ~l7 non-A non-B hepatitis virus polypeptide of the present ~
invention or a part thereof. The polypeptide or the -- -protein described above may be a complex of the non-A
non-B hepatitis virus polypeptide of the present invention or a part thereof with a carrier protein.
The complex may be prepared by using a coupling agent, if required. For example, glutaraldehyde, carbodiimide and maleimide active ester are usable therefor. As examples of the carrier protein, bovine serum albumin, thyroglobulin and hemocyanin may be cited. In a commonly employed case, the carrier protein is used in an amount of 1 to 5 times as much as the non-A non-B hepatitis virus polypeptide of the present invention or the part thereof.
As examples of the animal to be immunized, mouse, rat, rabbit and guinea pig may be cited. The inoculation may be effected by subcutaneous, intramuscular, intravenous or intraperitoneal administration. Before the inoculation, the antigen may be mixed with the complete Freund's adjuvant or the incomplete Freund's adjuvant and then administered. Usually, the inoculation is effected twice or more at intervals of 1 to 5 weeks. After sufficiently immunizing, the blood of the animal immunized is collected and the serum is separated to 2 1 2 ~q~

thereby give a polyclonal antibody. If necessarY.
purification may be effected to give the immunoglobulin fraction. -~
Antibody-producing cells are isolated as hybridomas which is prepared by fusing the antibody-producing cells obtained from the spleen or lymph nodes of the animal immunized with myeloma cells. As the myeloma cells, those originating in mouse, rat or human may be used. Although it is preferable that these myeloma cells have the same origin as that of the antibody-producing cells, those originating in different species can be fused with each other in some cases.
The cell fusion can be effected by a known method, for example, the method of Kohler and Milstein [see Nature, 2~, 495 (1975)]. As examples of the fusion promoter, polyethylene glycol and Sendai virus may be cited. The cell fusion can be usually carried out by reacting the antibody-producing cells with the myeloma cells at a ratio by cell count of about 1 : 1 to 10 : 1 at a temperature of 20 to 40 C (preferablY
30 to 37 C) for about 1 to 10 minutes by using polyethylene glycol (average molecular weight: 1,000 to 4,000) at a final concentration of about 20 to 50 %.

~ :~ 2 '~

For screening the antibody-producing hybridomas, various immunochemical methods can be employed. For example, the ELISA (enzyme-linked immnosorbent assay) with the use of a microplate coated with an immune antigen, the EIA (enzyme immunoassay) method with the use of a microplate coated with antiimmunoglobulin antibody and the Western blotting method with the use of a nitrocellulose membrane having an antigen in the form of a solid phase are usable therefor.
With respect to the antibody-producing hybridomas in wells which are confirmed to produce the antibody by the above described immunochemical method, cloning `~
is further effected by, for example, the limiting -~
dilution analysis to thereby select an objective clone(s). In the selection of the objective hybridomas and the growing the selected hybridomas, a medium for animal cells containing 10 to 20 ~O of fetal calf serum (for example, PRMI 1640) to which HAT
(hypoxanthine, aminopterin and thymidine) have been added is usually employed.
The clone thus obtained is intraperitoneally transplanted into a BALB/c mouse to which pristane has been administered. 10 to 14 days thereafter, the ascites fluid containing the monoclonal antibody at a high concentration is collected from the mouse !, ;A

- 21~2~

immunized, and used as a material for the purification of the antibody. Also, a culture, which is obtained by incubating the clone thus obtained, can be used as a material for the purification of the antibody. To recover the monoclonal antibody, a known method for the purification of immunoglobulin may be used. For example, it can be easily achieved by ammonium sulfate fractionation, PEG fractionation, ethanol fractionation, use of an anion exchanger and affinity chromatography.
These antibodies make it possible to identify and assay non-A non-B hepatitis virus antigen in a biological sample by a known immunological method.
Thus, these antibodies are usable as a diagnostic reagent for the non-A non-B hepatitis virus antigen.

(5) Gene analysis on non-A non-B hepatitis virus There have been reported 4 types of non-A non-B
hepatitis virus including the above-mentioned one reported by Houghton et al. (HCV-1), one reported by Kato (HCV-J), one reported by Okamoto et al. (HC-J6) and the virus of the present invention. As these facts clearly indicate, it is known that there are several subtypes of the non-A non-B hepatitis virus.
As discussed above, it is difficult to identify and distinguish these viral subtypes by 21~ 2~

immunologically assaying an antibody against the virus or an antigen polypeptide of this virus in a biological sample, though this method is useful for the diagnosis of the infection with this virus.
The DNA described in SEQ ID N0: 4 corresponds to a part of a region which encodes the structural ;~
protein of the virus, while the DNAs described in SEQ
ID N0: ~ and SEQ ID N0: 6 each corresponds to a part of a region which encodes the non-structural protein thereof. A comparison of the DNA base sequence of~ -~
each subtype of non-A non-B hepatitis virus with the DNA base sequences of the present invention shows a ~.-homology of 70 to 80 %. This fact indicates that the subtypes of the non-A non-B hepatitis virus can be -distinguished from each other by the gene analysis : .
with the use of a suitable oligonucleotide as a primer on the basis of the DNA base sequence of the corresponding part of the non-A non-B hepatitis virus. :
The oligonucleotide to be used as the primer comprises at least 6 bases which is contained in the DNA encoding the polypeptide of the present invention.
The number of the bases derived from the DNA encoding the polypeptide of the present invention in the .
oligonucleotide is preferably at least 8, still more preferably at least 10 to 12, and most preferably from 2~a27 15 to 25. That is, the base sequence of thP primer may be appropriately selected based on the base sequence (DNA
sequence) of the virus of each subtype.
The subtype of the non-A non-B hepatitis virus can be identified in the conventional manner by using an RNA prepared from a biological sample such as a plasma specimen as a template, amplifying a DNA by the RT-PCR
method with the use of an appropriate antisense primer, a sense primer, a reverse transcriptase and a thermostable DNA polymerase and then analyzing the DNA. The type of the virus can be identified by examining whether the DNA of the given size has been amplified or not by polyacrylamide gel or agarose gel electrophoresis. Further, the confirmation of the subtype can be made by using, as a probe, a labeled synthetic oligonucleotide which is derived from the virus of each subtype and of which sequence is present between two primers employed.
The structure of the amplified DNA may be analyzed by directly analyzing the base sequence from the PCR product. If necessary, it is recommended to integrate the DNA into an appropriate vector and then introduce this vector into an appropriate host followed by replication of the DNA. Thus the base sequence of the DNA can be analyzed.

0 ?'7 Further, the use of the non-A non-B hepatitis virus polypeptide of the present invention makes it possible to produce a vaccine which is usable as a medicine for preventing or treating non-A non-B
hepatitis.
The use of the DNA of the present invention. the RNA which is complementary thereto or an antigen polypeptide of the present invention enables the diagnosis of non-A non-B hepatitis, which makes it possible to prevent the infection with this virus via, for example, transfusion and to develop a vaccine and an antiviral agent therefor.
Examples The present invention will now be described in greater detail with reference to the following Examples which should not be considered to limit the scope Of the present invention. ~ -Example 1 Pr~?p~r~t~n of non-A non-R hep~t~t.~s v~r~ RNA fr~m p 1 ~q cmiq The plasmas of donors who had been judged as strongly positive for C100-3 an-tibody (2.00 >) by using an antibody detection reagent comprising a so-called viral antigen of Houghton et al. and the plasmas of patients with various hepatitises were :~. : . . - - . ~ . . . .
, , - - ~ -:: . -- 2~2~2'~

subjected to the identification of subtypes in accordance with the method disclosed in International Publication No. W092/18532 (published on Oct. 29, 1992), and corresponding thereto of European Patent Publication-A No. 580754 (published on Feb. 2, 1994).
Then, plasmas containing only the virus 2-22 as described in the International Publication No. W0 92/18532 were employed as a starting material to prepare the RNA.
To 1 ml of the plasma of a donor was added polyethylene glycol 4000 in such a manner as to give a final concentration of 4 %. After dissolution by stirring at 4 ~C for 90 minutes, the solution thus obtained was centrifugated at 14,000 rpm for 10 minutes to give a precipitate. To this precipitate were added 200 ~l of 6 M guanidine isothiocyanate, 2.1 ~l of ~-mercaptoethanol, 5 ~l of yeast t-RNA (10 mg/ml), 22 ~l of 2 M sodium citrate (pH 4.0) and 350 ~l of phenol/chloroform (5 : 2). The resulting mixture was stirred at room temperature for 1 minute, allowed to stand at 0 C for 15 minutes and then centrifuged at 14,000 rpm for 10 minutes. The aqueous layer of this reaction mixture was taken out and 350 ~l of phenol/chloroform (5 : 2) was added thereto.
After stirring at room temperature for 1 minute, the s. Q ~ ~

mixture was allowed to stand at 0 C for 15 minutes and centrifuged at 14,000 rpm for 10 minutes. The aqueous layer of the reaction mixture was taken out and extracted with phenol/chloroform again. To the aqueous layer taken therefrom was added 2-propanol in the same amount as the aqueous layer and the mixture thus obtained was allowed to stand at -20 C for 12 hours or longer. Then, it was centrifuged at 14,000 rpm for 20 minutes to give a precipitate. To this precipitate were added 85 ~l of 4 M guanidine isothio- -cyanate, 0.7 ~l of ~-mercaptoethanol, 15 ~l of 2 M
sodium citrate (pH 4.0) and 100 ~l of 2-propanol to effect isopropanol precipitation to thereby prepare an RNA.
Example 2 t~enç? ampl~f1c~tion hv RT-Pt'.R metho~l an(l st.rllct.ur~l ana 1 vs 1 ~:
20 ~l of the RNA solution thus obtained was centrifuged at 14,000 rpm for 10 minutes to thereby give a precipitate. This precipitate was washed with 70 % ethanol and dried. Next, 25 ~1 of a reagent mixture [10 mM Tris HCl (pH 8.3), 50 mM KCl, 4 mM
MgCl2, 1 mM DTT, 0.001 % gelatin], 2 ~1 of a solution of an antisense primer (10 ~M) in the 3'-downstream side as will be described hereinafter and 10 ~l of a 2~2~2i~

solution of a mixture of 4 deoxynucleotides [1.25 mY
portions of dATP, dGTP, dCTP and dTTP] were added thereto to give 37 ~l of a solution.
This solution was heated successively at 70 C
for 1 minute and at 55 C for 1 minutes and then cooled to 0 C. After adding 2 ~l (80 U) of RNase inhibitor (mfd. by Promega) and 1 ~l (200 U) of a reverse transciptase (mfd. by BRL), the mixture thus obtained was reacted at 37 C for 30 minutes. Then.
it was heated to 95 C and maintained at that temperature for 5 minutes to thereby inactivate the enzyme. To 4 ~l of this reaction mixture was added 44.2 ~1 of a PCR solution [10 mM Tris HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2, 0.001 % gelatin, dNTPs mixture each 125 ~M]. Then, 0.5 ~l of a solution of a primer (10 ~M) having a base sequence of the sense strand in the 5'-upstream side of the region to be subjected to the gene amplification and 0.3 ~l of a solution of a primer (10 ~M) having a base sequence of the antisense strand in the 3'-downstream side of the above-mentioned region were added to thereby give a solution of 49 ~l in the total volume.
The primers employed herein were those which had been synthesized on the basis of the DNA sequences of HC-J8, HC-J6, HCV-J and those identified during the L ~ 6 ~ " ~

Q 2 ~ .

experiment. The original viruses and base position numbers of these sense primers and antisense primers ~
and sequences of which structures are identified by -using these primers are listed in Tables 1 and 2 in accordance with the base numbers given in the sequence listing. In these Tables, J, J6 and J8 mean respectively HCV-J, HC-J6 and HC-J8 as described above, while Seq. 4, Seq. 5 and Seq. 6 mean respectively SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO:

6 which will be described hereinafter. To effect cloning, a sequence recognized by restriction enzymes Sma I and Bam HI was added to the 5'-side of some sense primers, while another sequence recognized by restriction enzymes Sal I and Pst I was added to the 3'-side of some antisense primers.

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Table 1 Primer Origin of the primer Base number of and base number of the sequence the sequence used identified with the primer ::
Sense J, 44-67 Antisense J. 192-170 Seq. 4, 1-102 Sense J8, 151-173 Antisense Seq.4,751-731 Seq. 4, 95-730 Sense J, 719-739 Antisense J8, 847-828 Seq. 4, 673-748 Sense Seq. 4,719-740 Antisense J8, 1344-1325 Seq. 4, 741-1245 Sense J6, 4058-4080 Antisense J8, 4299-4281 Seq. 5, 1-198 Sense Seq. 5,130-150 Antisense J8, 4924-4902 Seq. 5, 150-819 Sense Seq. 5,778-800 Antisense Seq. 5,1348-1328 Seq. 5, i800-1326 Sense J8, 5278-5299 Antlsense J8, 5463-5442 Seq. 5, 1218-1359 2 ~ . 7 Table 2 . .
Primer Origin of the Base number of the primer and base sequence identified number of the with the primer sequence used Sense J8,5398-5419 ~-Antisense J8,6169-6150 Seq.5, 1338-2067 Sense Seq. 5, 2017-2037 Antisense J8,6721-6700 Seq. 5, 2037-2617 Sense Seq. 5, 2560-2579 Antisense Seq. 5, 2808-2789 Seq. 5, 2579-2787 Sense J8,6768-6787 Antisense J8,7050-7029 Seq. 5, 2706-2946 Sense Seq. 5, 2895-2915 Antisense J8,7161-7141 Seq. 5, 2915-3058 Sense J8,7059-7078 Antisense Seq. 6, 26-5 Seq. 5, 2988-3633 ~ ;
Sense J8,7691-7013 Antisense J8,8756-8637 Seq. 6, 1-937 Sense J,8597-8617 Antisense J,9412-9401 Seq. 6, 900-1696 ~ 1 2 ~ 2 ~!

After adding 1 ~1 of Taq polymerase (0.5 U, mfd.
by Perkin-Elmer Cetus) to the solution described above, denaturation (94 C, 1 minute), annealing (45 C; 1 minute) and polymerization (72 C, 3 minutes) were repeated 30 times. To 4 ~1 of the reaction mixture thus obtained were further added 90 ~1 of the above-mentioned PCR solution, 1 ~1 of a solution of the sense primer, 1 ~1 of a solution of the antisense primer and 2 ~1 of Taq polymerase and then the DNA was ;
amplified by repeating denaturation (94 C, 1 minute), annealing (45 C, 1 minute) and polymerization (72 C, 3 minutes) 30 times.
Now the cloning of the gene product amplified by the RT-PCR method will be illustrated.
The amplified gene fragments were electrophoresed ~`
on a polyacrylamide gel (4 to 5 %) and stained with ethidium bromide. Then, a DNA of the target length was recovered. This DNA was cleaved with restriction enzymes Bam HI and Sal I and the resultant fragments were inserted into the Bam HI - Sal I site of phage vectors M13mpl8 and M13mpl9. Then, the gene was cloned by the conventional manner and the base sequence was analyzed.
The base sequence was identified by the dideoxy termination method with the use of M13 phages in the :-2~2~ 3, ) conventional manner. Based on the base sequences of the obtained clones, the overlapping parts were checked up. Thus, the base sequences represented by SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6 have been identified. Based on these sequences, it has been deduced that the amino acid sequences encoded thereby are those described in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3. SEQ ID NO: 1 corresponds to the structural region, while both SEQ ID NO: 2 and SEQ ID
NO: 3 correspond to the nonstructural regions.
Example 3 C~nf i rm~ t i ~n ~f ep i tope ~ite It was confirmed which site of the amino acid sequence of the non-A non-B hepatitis virus-constituting polYpeptide of the present invention described in SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO:
3 would be suitable as an epitope. First, polypeptides listed in Table 3 were synthesized based on the amino acid sequence of the present invention and screened.

212f~;Q2~ :

Table 3 .
Peptide Location of amino acid Positive ratio sequence in sequence (%) in plasmas listing of positive donors (n = 130) CL SEQ ID. N0:1, 8-40 100 M1 SEQ ID. N0:1, 4-24 94.6 M2aE SEQ ID. N0:1, 39-53 62.3 M2cE SEQ ID. N0:1, 59-74 52.3 M3E SEQ ID. N0:1, 101-120 31.5 57 SEQ ID. N0:2, 443-482 84.6 MllE SEQ ID. N0:2, 453-469 58.5 M51E SEQ ID. N0:2, 671-684 24.6 M15E SEQ ID. N0:2, 1009-1022 3.1 M17E SEQ ID. N0:2, 1071-1086 24.6 M32E SEQ ID. N0:3, 52-72 17.7 ;
' " ~ ' First, each 5.0 ~g/ml solution containing a synthetic peptide was pipetted into a well of a microplate in an amount of 100 ~l to thereby coat the well therewith. After blocking with 100 ~l of a blocking buffer (10 mM PBS, 1 % BSA, 0.1 % Tween 20, pH 7.4), 100 ~l portions of a reaction solution (50 mM
Tris, 1 mM EDTA, 0.1 % NaN~, 10 % NRS, pH 8.0) were added to the wells. Next, 20 ~l portions of the plasma of a donor was added thereto and the microplate was incubated at 37 C for 60 minutes. After the incubation, the wells were washed with a washing ::
buffer (10 mM PBS. 0.1 % BSA, 0.1 % Tween 20, pH 7.4) and then 100 ~l portions of alkaline phosphatase-:.

- 29 - ~
' .:~' .

2l21~,.n2,~ ., labeled anti-human IgG antibody were added to the wells.
After incubating at 37C for 60 minutes and washing with the washing buffer, 100 ~1 portions of a solution of an enzyme substrate (p-nitrophenyl phosphate 1.0 mg/ml, carbonate buffer, pH 9.8) was added to the wells. After reacting at 37C for 30 minutes, the absorbances of the reaction solutions in the wells were measured at a wavelength of 405 nm.
In the right column of Table 3, the positive ratios to the plasmas of donors (130 specimens), who had been judged as positive with a reagent of the second generation, i.e., a second generational anti HCV antibody detection reagent, thus determined are given. As Table 3 shows, it is confirmed that the positive ratios determined by using the peptides CL, Ml and 57 are respectively 100%, 94.6% and 84.6%. Thus it is proved that these 3 peptides are effective as an epitope. Namely, this result means that a reagent comprising a part of the amino acid sequence of the present invention as the epitope is effective in the diagnosis of non-A non-B hepatitis. Since a novel epitope is employed in the diagnostic reagent of the present invention, it is expected that the diagnostic reagent would exert a usefulness, which has never been achieved by the conventional ones, through 2~2~(~2~

the further accumulation of clinical test data. The amino acid sequences of the peptides CL, M1 and 57 are represented by SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID
NO 9, respectively. ~ .

~ . .,', ~ - 31 - ~

_ 212~ 02'~ -SEQUENCE LISTING

(2) iNFORMATlON FOR SEQ ID NO:1:
(ij SEQUENCE CHARACTERISTICS:
(A) LENGTH: 328 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (vi) ORIGINAL SOURCE:
(A) ORGANISM: non-A non-B hepatitis virus (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: ~ :

Met Ser Thr Asn Pro Lys Pro Gln Arg Lys Thr Lys Arg Asn Thr Asn 5 1 0 1 5 ' ~
Arg Arg Pro Gln Asp Yal Lys Phe Pro Gly Gly Gly Gln Val Val Gly Gly Val Tyr Leu Leu Pro Arg Arg Gly Pro Arg Leu Gly Val Arg Ala Thr Arg Lys Thr Ser Glu Arg Ser Gln Pro Arg Glu Arg Arg Gln Pro lle Pro Lys Asp Arg Arg Ser Thr Gly Lys Ser Trp Gly Lys Pro Gly TYr Pro Trp Pro Leu Tyr Gly Asn Glu Gly Cys Gly Trp Ala Gly Trp Leu Leu Ser Pro Arg Gly Ser Arg Pro Thr Trp Gly Pro Thr Asp Pro Arg His Arg Ser Arg Asn Leu Gly Lys Val lle Asp Thr Leu Thr Cys ,~. .
~', 115 120 125 ~.~

: .

212~ 27 Gly Phe Ala Asp Leu Met Gly Tyr l le Pro Val Val Gly Ala Pro Val Gly Gly Val Ala Arg Ala Leu Ala His Gly Val Arg Val Leu Glu Asp Gly Val Asn Tyr Ala Thr Gly Asn Leu Pro Gly Cys Ser Phe Ser I le Phe Leu Leu Ala Leu Leu Ser Cys Val Thr Val Pro Val Ser Ala Val Glu Val Arg Asn I le Ser Ser Ser Tyr Tyr Ala Thr Asn Asp Cys Ser 195 200 205 ~ ~:
Asn Asn Ser lle Thr Trp Gln Leu Thr Asn Ala Val Leu His Leu Pro Gly Cys Val Pro Cys Glu Asn Asp Asn Gly Thr Leu Arg Cys Trp lle Gln Val Thr Pro Asn Val Ala Val Lys Tyr Arg Gly Ala Leu Thr His 245 250 2S5 ~ .
Ser Leu Arg Thr His Val Asp Met I le Val Met Ala Ala Thr Val Cys ~ ~:
260 26S 270 ~;
Ser Ala Leu TYr Val G1Y Asp Ala Cys Gly Ala lle Met lle Ala Ser :

Gln Ala Phe lle lle Ser Pro Glu Arg His Asn Phe Thr Gln Glu Cys 290 295 300 .
Asn Cys Ser lle TYr Gln Gly Arg lle Thr G1Y His Arg Met Ala Trp 305 310 315 3~20 Asp Met Met Leu Asn Trp Ser Pro , ~ 2 1 2 ~ ~ 2 ~

(2) INFORMAT10N F0R SEQ ID NO:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1211 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single ~-(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (vi) ORIGINAL SOURCE:
(A) ORGANISM: non-A non-B hepatitis virus (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:

Tyr Ala Ser Gln Cly Tyr Lys Val Leu Val Leu Asn Pro Ser Val Ala 5 10 15 .
Ala Thr Leu Gly Phe Gly Ala Tyr Met Ser Lys Ala His Cly lle Asn Pro Asn lle Arg Thr Cly Val Arg Thr Val Thr Thr Cly Asp Pro lle Thr Tyr Ser Thr Tyr Gly Lys Phe Leu Ala Asp Gly Gly Cys Ser Ala Gly Ala Tyr Asp lle lle lle Cys Asp Glu Cys His Ala Val Asp Ser Thr Thr lle Leu GIY lle Gly Thr Val Leu Asp Gln Ala Glu Thr Ala Gly lle Arg Leu Val Val Leu Ala Thr Ala Thr Pro Pro Gly Thr Val Thr Thr Pro His Ser Asn lle Glu Glu Val Ala Leu Ser Nis Glu Gly Glu lle Pro Phe Tyr Gly Lys Ala lle Pro Leu Ala Ser lle Lys Gly 2~2~2 ~

Gly Arg His l,eu lle Phe Cys His Ser Lys Lys Lys Cys Asp Glu Leu -Ala Ala Ala Leu Arg Gly Met G1Y Val Asn Ala Val Ala Phe Tyr Arg : ::

Gly Leu Asp Val Ser Val I le Pro Thr Gln Gly Asp Val Val Val Val Ala Thr Asp Ala Leu Met Thr Gly Tyr Thr Gly Asp Phe Asp Ser Val l le Asp Cys Asn Val Arg Val Thr Gln l le Val Asp Phe Ser Leu Asp Pro Thr Phe Thr l le Thr l le Gln Thr Val Pro Cln Asp Ala Val Ser 225 230 235 240 .
Arg Ser Gln Arg Arg Gly Arg Thr Gly Arg Gly Arg Leu Gly lle Tyr Arg Tyr Val Ser Ser Gly Glu Arg Pro Ser Gly Met Phe Asp Ser Ala Val Leu Cys Glu Cys Tyr Asp Ala Gly Ala Ala Trp Tyr Glu Leu Thr Pro Ala Glu Thr Thr Val Arg Leu Arg Ala TYr Phe Asn Thr Pro Gly :
290 295 300 :
Leu Pro Val Cys Gln AsP His Leu Glu Phe Trp Glu Ala Val Phe Thr Gly Leu Thr His lle Asp Ala His Phe Leu Ser Gln Thr Lys Gln Gly ::

Gly Asp Asn Phe Ala TYr Leu Thr Ala Tyr Gln Ala Thr Val Cys Ala 340 345 350 ~ ~.
Arg Ala LYS Ala Pro Pro Pro Ser Trp Asp Val Met Trp Lys Cys Leu 2 ~ 2 ~

Thr Arg Leu LYS Pro Thr Leu Thr Gly Pro Thr Pro Leu Leu Tyr Arg Leu Gly Ala Val Thr Asn Glu Val Thr Leu Thr His Pro Val Thr Lys Tyr lle Ala Thr Cys Met Gln Ala Asp Leu Glu lle Met Thr Ser Thr -~
405 410 415 rp Val Leu Ala Gly Gly Val Leu Ala Ala Val Ala Ala Tyr Cys Leu Ala Thr Gly Cys lle Ser lle lle Gly Arg Leu His Leu Asn Asp Arg Val Val Val Ala Pro Asp Lys Glu lle Leu Tyr Glu Ala Phe Asp Glu Met Glu Glu Cys Ala Ser Lys Ala Ala Leu lle Glu Glu Gly Gln Arg 465 470 475 480 et Ala Glu Met Leu Lys Ser Lys lle Gln Gly Leu Leu Gln Gln Ala 485 490 495 hr Lys Gln Ala Gln Asp lle Gln Pro Ala lle Gln Ser Ser Trp Pro LYS Leu Glu Gln Phe Trp Ala Lys His Met Trp Asn Phe lle Ser Gly lle Gln Tyr Leu Ala GIY Leu Ser Thr Leu Pro Gly Asn Pro Ala Val 530 535 5~0 Ala Ser Met Met Ala Phe Ser Ala Ala Leu Thr Ser Pro Leu Pro Thr 545 550 555 560 er Thr Thr lle Leu Leu Asn lle Met Gly Gly Trp Leu Ala Ser Gln 565 570 575 le Ala Pro Pro Ala GIY Ala Thr Gly Phe Val Val Ser Gly Leu Val - 36 - :

~2~27 Gly Ala Ala Val Gly Ser I le Gly Leu Pro Asn I le Leu Val Asp Val Leu Ala Gly Tyr Gly Ala Gly lle Ser Gly Ala Leu Val Ala Phe Lys l le Met Ser Gly Glu Lys Pro Ser Val Glu Asp Val Val Asn Leu Leu 625 630 635 640 ro Ala l le Leu Ser Pro Gly Ala Leu Val Val Gly Val l le Cys Ala 645 650 655 la lle Leu Arg Arg His lle Gly Gln Gly Glu Gly Ala Val Gln Trp Met Asn Arg Leu lle Ala Phe Ala Ser Arg Gly Asn His Val Ala Pro Thr His Tyr Val Ala Glu Ser Asp Ala Ser Gln Arg Val Thr Gln Val Leu Ser Ser Leu Thr I le Thr Ser Leu Leu Arg Arg Leu His Ala Trp 705 710 715 720 le Thr Glu Asp Cys Pro Val Pro Cys Ser Gly Ser Trp Leu Arg Asp 725 730 735 le Trp Asp Trp Val Cys Ser I le Leu Thr Asp Phe Lys Asn Trp Leu Ser Ser LYS Leu Leu Pro LYS Met Pro Gly Leu Pro Phe Ile Ser Cys Gln LYS Gly Tyr Lys Gly Val Trp Ala Gly Thr Gly Val Met Thr Thr Arg Cys Pro Cys Gly Ala Asn lle Ser Gly llis Val Arg Met Gly Thr 785 790 795 800 et Arg I le Thr Gly Pro Lys Thr Cys Leu Asn Leu Trp Gln Gly Thr 2l2~a2~l -Phe Pro I le Asn Cys Tyr Thr Glu Gly Pro Cys Val Pro Lys Pro Pro 820 825 830 ro Asn Ty} Lys Thr Ala I le Trp Arg Val Ala Ala Ser Glu Tyr Val Glu lle Thr Arg His Gly Ser Phe Ser Tyr Val Thr Gly Leu Thr Thr Asp Asn Leu Lys Val Pro Cys Cln Val Pro Ala Pro Glu Phe Phe Ser 865 870 875 880 rp Val Asp Gly Val Gln I le His Arg Phe Ala Pro Thr Pro Gly Pro 885 890 895 he Phe Arg Asp Glu Val Ser Phe Thr Val Gly Leu Asn Ser Phe Val 900 905 910 al Gly Ser Gln Leu Pro Cys Asp Pro Glu Pro Asp Thr Glu Val Leu Ala Ser Met Leu Thr Asp Pro Ser His lle Thr Ala Glu Ala Ala Ala Arg Arg Leu Ala Arg Gly Ser Pro Pro Ser Gln Ala Ser Ser Ser Ala 945 950 955 960 er Gln Leu Ser Ala Pro Ser Leu Lys Ala I le Cys Ala Ser His Lys 965 970 975 et Ala Tyr Asp CYS Asp Met Val Asp Ala Asn Leu Phe Met Gly Gly 980 985 990 sp Val Thr Arg l le Glu Ser Asp Ser Lys Val l le Val Leu Asp Tyr 995 1000 1005 :
Leu Asp Ser Met Thr Glu Thr Glu Asp Asp Arg Glu Pro Ser l le Pro Ser Glu Tyr Leu l le LYS Arg Lys Arg Phe Pro Val Ala Val Pro Ser 212~Q2 i/

Trp Ala His Pro Asp Tyr Asn Pro Pro Leu Val Glu Thr Trp Lys Arg Pro Gly Tyr Glu Pro Pro Thr Val Leu Gly Cys Ala Leu Pro Pro Thr 1060 1065 1070 :
Pro Gln Ala Pro Val Pro Pro Pro Arg Arg Arg Arg Ala Lys Val Leu Thr Gln Asp Asn Val Glu Gly Val Leu Arg Glu Met Ala Asp Lys Val 1090 1095 1100 ':' '~
Leu Ser Ser Leu Arg Gly Cys Asn Asp Thr Gly His Ser Thr Glu Met 1105 1110 lllS 1120 Asp Thr Gly Gly Asp Ser Val Gln Gln Pro Ser Asp Glu Thr Thr Ala : ~.

Ser Glu Glu Gly Ser Leu Ser Ser Met Pro Pro Leu Glu Gly Glu Pro 1140 1145 1150 :-Gly Asp Pro Asp Leu Glu Phe Gly Gln Ala Gly Ser Ala Pro Pro Ser ~ -Glu Glu Glu Gly Glu Val I le Asp Ser Asp Ser Lys Ser Trp Ser Thr Val Ser Asp Gln Glu Asp Ala Val I le Cys Cys Ser Met Ser Tyr Ser 1185 1190 ll9S 1200 Trp Thr Gly Ala Leu l le Thr Pro Cys Gly Pro -- 39 - :

2~7 (2) INFORMATION FOR SEQ ID NO:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: S47 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (vi) ORIGINAL SOURCE:
(A) ORGANISM: non-A non-B hepatitis virus (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:
la Ser Leu Arg Ala Lys Lys Val Thr Phe Asp Arg Val Gln Val L~eu 15 sp Ala His TYr Asp Ser Val Leu Gln Asp Val Lys Arg Ala Ala Ser Lys Val Ser Ala Arg Leu Leu Ser Val Glu Glu Ala Cys Ala Leu Thr Pro Pro His Ser Ala Lys Ser Leu Tyr Gly Phe Gly Ala Lys Clu Val Arg Ser Leu Ser Arg Arg Ala Val Asn His lle Arg Ser Val Trp Glu 6S 70 75 80 sp Leu Leu Clu Asp Gln His Thr Pro lle Asn Thr Thr lle Met Ala 95 ys Asn Glu Val Phe Cys lle Asp Pro Ala Lys Gly Gly Arg Lys Pro Ala Arg Leu lle Val TYr Pro Asp Leu Gly Val Arg Val Cys Glu Lys Met Ala Leu Tyr Asp lle Ala Gln Lys Leu Pro Lys Ala lle Met Gly ~ 2l' Q2 l Pro Ser TYr Gly Phe Gln Tyr Ser Pro Ala Glu Arg Val Asp Phe Leu Leu LYS Ala Trp Gly Ser Lys Lys Asp Pro Met Gly Phe Ser Tyr Asp Thr Arg Cys Phe Asp Ser Thr Val Thr Glu Arg Asp I le Arg Thr Glu 180 185 190 :.
Glu Ser lle Tyr Gln Ala Cys Ser Leu Pro Gln Glu Ala Arg Thr Val lle His Ser Leu Thr Glu Arg Leu Tyr Val Gly Gly Pro Met Thr Asn 210 215 220 ~ :
Ser Lys Gly Gln Ser Cys Gly TYr Arg Arg CYS Arg Ala Ser Gly Val -~

Phe Thr Thr Ser Met Gly Asn Thr Met Thr Cys Tyr I le Lys Ala Leu Ala Ala Cys Lys Ala Ala Gly lle Lys Asp Pro lle Met Leu Val Cys Gly Asp Asp Leu Val Val lle Ser Glu Ser Gln Gly Asn Glu Glu Asp -Glu Arg Asn Leu Arg Ala Phe Thr Glu Ala Met Thr Arg Tyr Ser Ala -Pro Pro Gly Asp Pro Pro Arg Pro Glu TYr Asp Leu Glu Leu lle Thr Ser Cys Ser Ser Asn Val Ser Val Ala Leu Asp Pro Arg Gly Arg Arg Arg TYr Tyr Leu Thr Arg Asp Pro Thr Thr Pro l le Ser Arg Ala Ala Trp Glu Thr Val Arg His Ser Pro Val Asn Ser Trp Leu Gly Asn l le ~2'~

lle Gln Tyr Ala Pro Thr lle Trp Val Arg Met Val lle Met Thr His Phe Phe Ala I le Leu Leu Ala Gln Asp Thr Leu Asn Gln Asn Leu Asn Phe Glu Met TYr Gly Ala Val Tyr Ser Val Asn Pro Leu Asp Leu Pro Ala lle lle Glu Arg Leu His Gly Leu Asp Ala Phe Ser Leu His Thr Tyr Ser Pro His Glu Leu Ser Arg Val Ala Ala Thr Leu Arg LYS Leu :

Gly Ala Pro Pro Leu Arg Ala Trp Lys Ser Arg Ala Arg Ala Val Arg --450 455 460 ::
Ala Ser Leu I le Ala Gln Gly Gly Arg Ala Ala l le Cys Gly Arg Tyr Leu Phe Asn Trp Ala Val Lys Thr Lys Leu Lys Leu Thr Pro Leu Pro 485 490 495 ~:
Glu Ala Ala Arg Leu Asp Leu Ser Gly Trp Phe Thr Val Gly Ala Gly Gly Gly Asp l le Phe His Ser Val Ser Arg Ala Arg Pro Arg Leu Leu Leu Leu CYS Leu Leu Leu Leu Ser Val Gly Val Gly I le Phe Leu Leu Pro A I a Arg , . ' .

; Q ~ 7 (2) INFORMATION FOR SEQ ID NO:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1246 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA to genomic RNA
(vi) ORIGINAL SOURCE:
(A) ORGANIS,M: non-A non-8 hepatitis virus (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: :
-Met Ser Thr Asn Pro Lys Pro Gln Arg Lys 5 10 ~ ' Thr Lys Arg Asn Thr Asn Arg Arg Pro Gln Asp Val Lys Phe Pro Gly 15 20 25 ~ .

Gly GIY Gln Val Val Gly Gly Val Tyr Leu Leu Pro Arg Arg Gly Pro 30 35 40 ~ ;

Arg Leu Gly Val Arg Ala Thr Arg Lys Thr Ser Glu Arg Ser Gln Pro :

212`~ ~2~

Arg Glu Arg Arg Gln Pro lle Pro Lys Asp Arg Arg Ser Thr Gly Lys Ser Trp Gly Lys Pro Gly Tyr Pro Trp Pro Leu Tyr Gly Asn Glu Gly Cys Gly Trp Ala Cly Trp Leu Leu Ser Pro Arg Gly Ser Arg Pro Thr Trp Gly Pro Thr Asp Pro Arg His Arg Ser Arg Asn Leu Gly LYS Val ATC GAT ACC CTT ACG TGT GGT TTT GCC GAC CTC ATG GGG TAC ATC CCT 676 :
lle Asp Thr Leu Thr Cys Gly Phe Ala Asp Leu Met Gly Tyr lle Pro :

Val Val Gly Ala Pro Val Gly Gly Val Ala Arg Ala Leu Ala His Gly Val Arg Val Leu Glu Asp Gly Val Asn Tyr Ala Thr Gly Asn Leu Pro GGT TGC TCC TTT TCT ATC TTC TTA CTT GCT CTT CTG TCG TGC GTT ACA 820 . :
Gly Cys Ser Phe Ser lle Phe Leu Leu Ala Leu Leu Ser Cys Val Thr Val Pro Val Ser Ala Val Glu Val Arg Asn lle Ser Ser Ser Tyr TYr 2~Q~7 Ala Thr Asn Asp Cys Ser Asn Asn Ser lle Thr Trp Gln Leu Thr Asn Ala Val Leu His Leu Pro Gly Cys Val Pro Cys Glu Asn Asp Asn Gly 220 225 230 . :

Thr Leu Arg Cys Trp lle Gln Val Thr Pro Asn Val Ala Val Lys Tyr .-CGC GGC GCA CTC ACC CAT AGC CTG CGA ACA CAT GTC GAC ATG ATC GTA 1060 ;
Arg Gly Ala Leu Thr His Ser Leu Arg Thr His Val Asp Met lle Val Met Ala Ala Thr Val Cys Ser Ala Leu Tyr Val Gly Asp Ala Cys Gly Ala lle Met lle Ala Ser Gln Ala Phe lle lle Ser Pro Glu Arg His Asn Phe Thr Gln Glu Cys Asn Cys Ser lle Tyr Gln Gly Arg lle Thr Gly His Arg Met Ala Trp Asp Met Met Leu Asn Trp Ser Pro 21~ r~

(2) INFORMATION POR SEQ ID NO:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 3633 base pairs -(B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA to genomic RNA
(vi) ORIGINAL SOURCE:
(A) ORGANISM: non-A non-B hepatitis virus (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:

Tyr Ala Ser Gln Gly TYr Lys Val Leu Val Leu Asn Pro Ser Val Ala 5 1 0 1 5 :. ' ' Ala Thr Leu Gly Phe Gly Ala Tyr Met Ser Lys Ala His Gly lle Asn Pro Asn lle Arg Thr GIY Val Arg Thr Val Thr Thr Gly Asp Pro lle Thr Tyr Ser Thr Tyr Gly LYS Phe Leu Ala Asp Gly Gly Cys Ser Ala .; ::

GGC GCC TAT GAC ATC ATC ATA TGC GAC GAA TGT CAT GCA GTG GAC TCT 240 .
Gly Ala Tyr Asp lle lle lle Cys Asp Glu CYS His Ala Val Asp Ser Thr Thr lle Leu Gly lle Gly Thr Val Leu Asp Gln Ala Glu Thr Ala 85 90 95 :

12~Q2~

Gly lle Arg Leu Val Val Leu Ala Thr Ala Thr Pro Pro GIY Thr Val ACA ACT CCC CAT AGT AAC ATA GAG GAG GTG GCT CTT AGT CAT GAA GGC 384 - .
Thr Thr Pro His Ser Asn lle Glu Glu Val Ala Leu Ser His Glu Gly Glu lle Pro Phe Tyr Gly Lys Ala lle Pro Leu Ala Ser lle Lys Gly GGC AGA CAC CTG ATC TTC TGC CAT TCA AAG AAG AAA TGC GAT GAA CTC 480 ;
Gly Arg His Leu lle Phe Cys His Ser Lys Lys Lys Cys Asp Glu Leu Ala Ala Ala Leu Arg Gly Met Gly Val Asn Ala Val Ala Phe Tyr Arg Gly Leu Asp Val Ser Val lle Pro Thr Gln Gly Asp Val Val Val Val Ala Thr Asp Ala Leu Met Thr Gly Tyr Thr Gly Asp Phe Asp Ser Val .~ ;

ATT GAC TGC AAC GTT CGA GTC ACT CAG ATT GTT GAC TTT AGC CTA GAT 672 ;
lle Asp Cys Asn Val Arg Val Thr Gln lle Val Asp Phe Ser Leu Asp , . 210 . 215 220 Pro Thr Phe Thr lle Thr lle Gln Thr Val Pro Gln Asp Ala Val Ser - . - . : . . .

i Q ?~ ~ -Arg Ser Gln Arg Arg Gly Arg Thr GIY Arg Gly Arg Leu Gly l le Tyr Arg Tyr Val Ser Ser Gly Glu Arg Pro Ser Gly Met Phe Asp Ser Ala :

Val Leu Cys Glu Cys Tyr Asp Ala Gly Ala Ala Trp Tyr Glu Leu Thr Pro Ala Glu Thr Thr Val Arg Leu Arg Ala Tyr Phe Asn Thr Pro G:ly Leu Pro Val Cys Gln Asp His Leu Glu Phe Trp Glu Ala Val Phe Thr ~ . .

Gly Leu Thr His lle Asp Ala His Phe Leu Ser Gln Thr Lys Gln Gly ~.

Gly Asp Asn Phe Ala TYr Leu Thr Ala Tyr Gln Ala Thr Val Cys Ala AGG G(;A AAG GCC CCT CCA CCT TCG TGG GAC GTG ATG TGG AAG TGT CTA 1104 Arg Ala Lys Ala Pro Pro Pro Ser Trp Asp Val Met Trp Lys Cys Leu ACT AGG CTC AAA CCT ACA CTG ACT GGT CCT ACC CCC CTC CTG TAC CGC 1152 : .
Thr Arg Leu Lys Pro Thr Leu Thr Gly Pro Thr Pro Leu Leu Tyr Arg ; ~ ~
370 375 380 : ;
' .

2 ~

Leu Gly Ala Val Thr Asn Glu Val Thr Leu Thr His Pro Val Thr Lys Tyr lle Ala Thr Cys Met Gln Ala Asp Leu Glu lle Met Thr Ser Thr Trp Val Leu Ala Gly Gly Val Leu Ala Ala Val Ala Ala Tyr Cys Leu ::~

Ala Thr Gly Cys lle Ser lle lle Gly Arg Leu His Leu Asn Asp Arg 435 440 445 :~
GTG GTT GTG GCC CCT GAC AAG GAG ATC TTA TAC GAG GCC TTT GAT GAG 1392 :
Val Val Val Ala Pro Asp Lys Glu lle Leu Tyr Glu Ala Phe Asp Glu Met Glu Glu Cys Ala Ser Lys Ala Ala Leu lle Glu Glu Gly Gln Arg Met Ala Glu Met Leu LYS Ser Lys lle Gln Gly Leu Leu Gln Gln Ala Thr Lys Gln Ala Gln Asp lle Gln Pro Ala lle Gln Ser Ser Trp Pro Lys Leu Glu Gln Phe Trp Ala Lys His Met Trp Asn Phe lle Ser Gly . ~, -, : ........... . ~ .. ~
- . . : : :: , ;:: .. . .:

2 ~ 7 lle Gln Tyr Leu Ala Gly Leu Ser Thr Leu Pro Gly Asn Pro Ala Val Ala Ser ,\let ?det Ala Phe Ser Ala Ala Leu Thr Ser Pro Leu Pro Thr Ser Thr Thr I le Leu Leu Asn I le Met Gly Gly Trp Leu Ala Ser Gln 565 570 575 :

lle Ala Pro Pro Ala GIY Ala Thr Gly Phe Val Val Ser Gly Leu Val GGG GCG GCC GTC GGG AGT ATA GGT CTA CCT AAC ATA CTG GTG GAC GTC 1824 :
Gly Ala Ala Val Gly Ser I le Gly Leu Pro Asn I le Leu Val Asp Val Leu Ala Gly Tyr Gly Ala Gly lle Ser Gly Ala Leu Val Ala Phe Lys ATC ATG AGC GGC GAG AAG CCC TCG GTA GAA GAT GTT GTG AAT CTC CTG 1920 ~.
I le Met Ser Gly Glu LYS Pro Ser Val Glu Asp Val Val Asn Leu Leu Pro Ala I le Leu Ser Pro GIY Ala Leu Val Val GIY Val I le Cys Ala GCG ATC TTG CGC CGC CAC ATC GGT CAG GGA GAG GGG GCG GTT CAG TGG 2016 `
Ala I le Leu Arg Arg His I le Gly Gln Gly Glu Gly Ala Val Gln Trp 2 ~ 2 ~

Met Asn Arg Leu lle Ala Phe Ala Ser Arg Gly Asn His Val Ala Pro Thr His Tyr Val Ala Glu Ser Asp Ala Ser Gln Arg Val Thr Gln Val TTC AGT TCA CTC ACA ATT ACC AGC TTA CTT AGG AGA CTA CAT GCC TGG 2160 .
Leu Ser Ser Leu Thr lle Thr Ser Leu Leu Arg Arg Leu His Ala Trp lle Thr Glu Asp Cys Pro Val Pro Cys Ser Gly Ser Trp Leu Arg Asp 725 730 735 :

lle Trp Asp Trp Val Cys Ser lle Leu Thr Asp Phe Lys Asn Trp Leu 740 745 750 :
TCT TCA AAA CTG CTC CCC AAA ATG CCT GGC CTC CCC TTT ATC TCT TGT 2304 : .
Ser Ser Lys Leu Leu Pro Lys Met Pro Gly Leu Pro Phe lle Ser Cys Gln Lys Gly Tyr Lys Gly Val Trp Ala Gly Thr G1Y Val Met Thr Thr Arg Cys Pro Cys Gly Ala Asn lle Ser Gly His Val Arg Met Gly Thr Met Arg lle Thr Gly Pro Lys Thr Cys Leu Asn Leu Trp Gln Gly Thr 2~2~2~1 Phe Pro l le Asn Cys Tyr Thr Glu Gly Pro Cys Val Pro LYs Pro Pro Pro Asn Tyr Lys Thr Ala lle Trp Arg Val Ala Ala Ser Glu Tyr Val 835 840 845 ` ~:-Glu lle Thr Arg His Gly Ser Phe Ser Tyr Val Thr Gly Leu Thr Thr GAC AAC CTT AAG GTC CCT TGC CAG GTA CCA GCT CCA GAA TTC TTC TCT 2640 : .
Asp Asn Leu Lys Val Pro Cys Gln Val Pro Ala Pro Glu Phe Phe Ser ;;
865 870 875 880 ~ :
TGG GTG GAT GGG GTG CAG ATA CAC CGA TTC GCC CCC ACT CCA GGT CCC 2688 .
Trp Val Asp Gly Val Gln l le His Arg Phe Ala Pro Thr Pro Gly Pro Phe Phe Ar~ Asp Glu Val Ser Phe Thr Val Gly Leu Asn Ser Phe Val Val Gly Ser Gln Leu Pro Cys Asp Pro Glu Pro Asp Thr Glu Val Leu :

:.
GCC TCC ATG CTG ACA GAT CCG TCC CAC ATT ACA GCG GAG GCG GCA GCT 2832 : .
Ala Ser Met Leu Thr Asp Pro Ser His lle Thr Ala Glu Ala Ala Ala Arg Arg Leu Ala Arg Gly Ser Pro Pro Ser Gln Ala Ser Ser Ser Ala : , - 52 - ~

212~?J7 Ser Gln Leu Ser Ala Pro Ser Leu Lys Ala lle Cys Ala Ser His Lys 965 970 975 :

Met Ala Tyr Asp Cys Asp Met Val Asp Ala Asn Leu Phe Met Gly Gly GAT GTG ACC CGG ATC GAG TCT GAC TCT AAG GTG ATT GTT CTC GAC TAT 3024 :
Asp Val Thr Arg lle Glu Ser Asp Ser Lys Yal lle Val Leu Asp Tyr Leu Asp Ser Met Thr Glu Thr Glu Asp Asp Arg Glu Pro Ser l le Pro Ser Glu Tyr Leu l le Lys Arg Lys Arg Phe Pro Val Ala Val Pro Ser Trp Ala His Pro Asp Tyr Asn Pro Pro Leu Val Glu Thr Trp Lys Arg Pro Gly Tyr Glu Pro Pro Thr Val Leu Gly Cys Ala Leu Pro Pro Thr Pro Cln Ala Pro Val Pro Pro Pro Arg Arg Arg Arg Ala Lys Val Leu Thr Gln Asp Asn Val Glu Gly Val Leu Arg Glu Met Ala Asp Lys Val `7 ~ S `

212`~ ~2~

Leu Ser Ser Leu Arg Gly Cys Asn Asp Thr Gly His Ser Thr Glu Met GAT ACC GGG GGA GAC AGC GTC CAG CAG CCC TCT GAC GAG ACT ACT GCT 3408 :
Asp Thr Gly Gly Asp Ser Val Gln Gln Pro Ser Asp Glu Thr Thr Ala Ser Glu Glu Gly Ser Leu Ser Ser Met Pro Pro Leu Glu Gly Glu Pro ~:
1140 1145 llS0 Gly Asp Pro Asp Leu Glu Phe Gly Gln Ala Gly Ser Ala Pro Pro Ser ~.

GAG GAG GAG GGC GAG GTC ATT GAT TCG GAC TCT AAG TCG TGG TCT ACA 3552 ~ :
Glu Glu Glu Gly Glu Val lle Asp Ser Asp Ser Lys Ser Trp Ser Thr :~
1170 1175 1180 ~:-Val Ser Asp Gln Glu Asp Ala Val lle Cys Cys Ser Met Ser Tyr Ser Trp Thr Gly Ala Leu lle Thr Pro Cys Gly Pro -~,12~

(2) INFORMATION FOR SEQ ID NO:6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1696 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA to genomic RNA
(vi) ORIGINAL SOURCE:
(A) ORGANISM: non-A non-B hepatitis virus (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:

Ala Ser Leu Arg Ala Lys Lys Val Thr Phe Asp Arg Val Gln Val Leu Asp Ala His Tyr Asp Ser Val Leu Gln Asp Val Lys Arg Ala Ala Ser Lys Val Ser Ala Arg Leu Leu Ser Val Glu Glu Ala Cys Ala Leu Thr Pro Pro His Ser Ala 1,YS Ser Leu Tyr Gly Phe Gly Ala Lys Glu Val Arg Ser Leu Ser Arg Arg Ala Val Asn His lle Arg Ser Val Trp Glu GAC CTC CTG GAA GAC CAA CAT ACT CCA ATT AAC ACA ACC ATC ATG GCC 288 : :
Asp Leu Leu Glu AsP Gln His Thr Pro lle Asn Thr Thr lle Met Ala ~ ~ 2 ' ~

: :.

Lys Asn Glu Val Phe Cys lle Asp Pro Ala Lys Gly Gly Arg Lys Pro 100 105 110 ' , Ala Arg Leu lle Val Tyr Pro Asp Leu Gly Val Arg Val Cys Glu Lys Met Ala Leu Tyr Asp lle Ala Gln Lys Leu Pro Lys Ala lle Met Gly Pro Ser Tyr Gly Phe Gln Tyr Ser Pro Ala Glu Arg Val AsP Phe Leu Leu Lys Ala Trp Gly Ser Lys LYS ASP Pro Met Gly Phe Ser Tyr Asp Thr Arg Cys Phe Asp Ser Thr Val Thr Glu Arg Asp lle Arg Thr Glu 180 185 190 :;

Glu Ser lle Tyr Gln Ala Cys Ser Leu Pro Gln Glu Ala Arg Thr Val ATA CAC TCG CTC ACT GAG AGA CTT TAC GTA GGA GGG CCC ATG ACA AAC 672 ~ ~ .
lle His Ser Leu Thr Glu Arg Leu Tyr Val Gly Gly Pro Met Thr Asn 210 ~15 220 Ser Lys Gly Gln Ser Cys Gly Tyr Arg Arg Cys Arg Ala Ser Gly Val 212'~ Q~'~

Phe Thr Thr Ser Met Gly Asn Thr Met Thr Cys Tyr lle Lys Ala Leu Ala Ala Cys Lys Ala Ala Gly lle Lys Asp Pro lle Met Leu Val Cys 260 26~ 270 Gly Asp Asp Leu Val Val lle Ser Glu Ser Gln Gly Asn Glu Glu Asp Glu Arg Asn Leu Arg Ala Phe Thr Glu Ala Met Thr Arg Tyr Ser Ala Pro Pro Gly Asp Pro Pro Arg Pro Glu Tyr Asp Leu Glu Leu lle Thr Ser Cys Ser Ser Asn Val Ser Val Ala Leu Asp Pro Arg Gly Arg Arg Arg Tyr Tyr Leu Thr Arg Asp Pro Thr Thr Pro lle Ser Arg Ala Ala Trp Glu Thr Val Arg His Ser Pro Val Asn Ser Trp Leu Gly Asn lle 355 360 365 ~:

lle Gln Tyr Ala Pro Thr lle Trp Val Arg Mst Val lle Met Thr His - 57 - .: .

... ., . . . , . ~ . . . . . . . . . .

212~.Q,~r,?

Phe Phe Ala l le Leu Leu Ala Gln Asp Thr Leu Asn Gln Asn Leu Asn Phe Glu Met Tyr Gly Ala Val Tyr Ser Val Asn Pro Leu Asp Leu Pro ~05 410 415 :~
GCC ATA ATT GAA AGG TTA CAT GGG CTT GAT GCC TTT TCA CTG CAC ACT 1296 ~ :
Ala lle lle Glu Arg Leu His Gly Leu Asp Ala Phe Ser Leu His Thr :
420 425 430 .

Tyr Ser Pro His Glu Leu Ser Arg Val Ala Ala Thr Leu Arg Lys L:eu Gly Ala Pro Pro Leu Arg Ala Trp Lys Ser Arg Ala Arg Ala Val Arg Ala Ser Leu l le Ala Gln Gly Gly Arg Ala Ala I le Cys GIY Arg Tyr Leu Phe Asn TrP Ala Val Lys Thr Lys Leu Lys Leu Thr Pro Leu Pro : ~
485 490 495 .;:

Glu Ala Ala Arg Leu Asp Leu Ser Gly Trp Phe Thr Val Gly Ala Gly GGG GGC GAC ATC TTT CAC AGC GTG TCG CGT GCC CGA CCC CGC TTA TTA 1584 :
Gly Gly Asp l le Phe His Ser Val Ser Arg Ala Arg Pro Arg Leu Leu 2~ .a2~

Leu Leu Cys Leu Leu Leu Leu Ser Val Gly Val Gly ile Phe Leu Leu Pro Ala Arg (2) INFORMATION POR SEQ ID NO:7:
(i) SEQUENCE CHARACTERISTICS: ~
(A) LENGTH: 33 amino acids ::
(B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide :
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:

Gln Arg LYS Thr i,Ys Arg Asn Thr Asn Arg Arg Pro Gln Asp Val Lys Phe Pro GIy GIY Cly Gln Val Val Gly Gly Val Tyr Leu Leu Pro Arg Arg 2 ~ 2 ~ ~

(2) INFORMATION FOR SEQ ID NO:8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:

Asn Pro LYS Pro Gln Arg Lys Thr Lys Arg Asn Thr Asn Arg Arg Pro Gln Asp Val Lys Phe : ,' . '.:
' ~ ' (2) INFORMATION FOR SEQ ID NO:9: ~ :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 40 amino acids (B) TYPE: amino acid .~:
(D) TOPOLOQY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:

! Leu His Leu Asn AsP Arg Val Val Val Ala Pro Asp LYS Glu lle Leu Tyr Glu Ala Phe AsP Glu Met Glu Glu Cys Ala Ser LYS Ala Ala Leu : :
lle Glu Glu Gly Gln Arg Met Ala 35 ~0 -:

F ~ ; ;

2~ Q2~

(2) INFORMATION FOR SEQ ID NO:10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:

Arg Arg Gly Pro Arg Leu Gly Val Arg Ala Thr Arg Lys Thr Ser :
'~

(2) INFORMATION FOR SEQ ID NO:11:
(i) SEQUENCE CHARACTERlSTlCS~
(A) LENGTH: 16 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: -Arg Glu Arg Arg Gln Pro Ile Pro Lys Asp Arg Arg Ser Thr Gly Lys 21~ 3~

(2) INFORMATION FOR SEQ ID NO:12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide :~
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: ~ :

Arg Gly Ser Arg Pro Thr Trp Gly Pro Thr Asp Pro Arg His Arg Ser Arg Asn Leu Gly (2) INFORMATION FOR SEQ ID NO:13:
(i) SEQUENCE CNARACTERISTICS:
(A) LENGTH: 17 amino acids -;
(B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:

Pro Asp LYS Glu Ile Leu Tyr Glu Ala Phe Asp Glu Met Glu Glu Cys Ala 2 1 ~

(2) INFORMATION FOR SEQ ID NO:14:
(i) SEQUENCE CHARACTERISTICS: ~-(A) LENCTH: 14 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide -(xi) SEQUENCE DESCRIPTION: SEQ ID NO:14: ~:

Gln Trp Met Asn Arg Leu lle Ala Phe Ala Ser Arg Gly Asn (2) INFORMATION FOR SEQ ID NO:15:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids (B) TYPE: amino acid .
(D) TOPOLOGY: linear :
(ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:

Leu Asp Ser Met Thr Glu Thr Glu Asp Asp Arg Glu Pro Ser - 6~ --- 2124~7 (2) INFORMATION FOR SEQ ID NO:16:
(i) SEQUENCE CHARACTERISTICS: .
(A) LENCTH: 16 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide ~ -(xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:

Pro Thr Pro Gln Ala Pro Val Pro Pro Pro Arg Arg Arg Arg Ala Iys ' : .
(2) INFORMATION FOR SEQ ID NO:17:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:

Ser Ala LYS Ser Leu Tyr Gly Phe Gly Ala Lys Glu Val Arg Ser Leu Ser Arg Arg Ala Val '.'' .,.. ~ .. .. ~ - ~

~2~ Q2'7 (2) INFORMATION FOR SEQ ID NO:18:
(i) SEQUENCE CHARACTERI STICS:
(A) LENGTH: 24 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18: :.
' TACTGTCTTC ACGCAGAAAG CGTC 24 .

(2) INFORMATION FOR SEQ ID NO:l9:
(i) SEQUENCE CHARACTERI STICS:
(A) LENGTH: 23 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single :
(D) TOPOLOGY: linear : ::
(ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRI PTI ON: SEQ ID NO:19:
, . ,, ' GATCCAAGAA AGGACCCGGT CGT 23 ~
~,.

- 65 - :
- .~' .

2l~a2t~ .

(2) INFORMATION FOR SEQ ID NO:20:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:

(2) INFORMATION FOR SEQ ID NO:21:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 33 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:

GGTACCGTCG ACATTTACCC CGTCTTCCAG GAC 33 ;:

'. ' :'' , :" ''' ",,''''~ ' ' '` ", :

2~2~

-(2) INFORMATION FOR SEQ ID NO:22: .
(i~ SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32 base pairs --~
(B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:

(2) INFORMATION FOR SEQ ID NO:23:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32 base pairs (B) TYPE: nucleic acid .
(C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) ;
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:23: ~ :~

GGTACCGTCG ACAAATTCCC TGTTGCGTAA TT 32 ~:

- 67 - ~
: ~ . ' ~,.~,,, . .. ~. . .: , . : . .

`i .' ': i . -':.' ',:' ", ~ ' ,' '': ' ` '. ` . ' : ': '' ` . ' ~ ' `: , ` . :' . : '' 2 ~

(2) INFORMATION FOR SEQ ID NO:24:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 33 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:
:

, ~:

(2) INFORMATION FOR SEQ ID NO:25:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32 base pairs (B) TYPE: nucleic acid :~
(C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:

- 2:~2.~Q2~

(2) INFORMATION FOR SEQ ID NO:26:
(i) SEQUENCE CHARACTERISTICS:
(A) LENCTH: 35 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single .
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:

(2) INFORMATION FOR SEQ ID NO:27:
(i) SEQUENCE CHARACTERISTICS:
(A) LENCTH: 32 base pairs ~: `
(B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:

::
.

2 1 ~ 7 (2) INFORMATION FOR SEQ ID NO:28:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 34 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:

(2) INFORMATION FOR SEQ ID NO:29:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 34 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:

~2~

(2) INFORMATION FOR SEQ ID NO:30:
(i) SEQUENCE CHARACTERISTICS: ~-(A) LENGTH: 34 base pairs (B) TYPE: nucleic acid .
(C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) ~::
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:30: :~
, '; -:

(2) INFORMATION FOR SEQ ID NO:31: ~
(i) SEQUENCE CHARACTERISTICS: ..
(A) LENGTH: 32 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single - `
(D) TOPOLOGY: linear `
(ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:

2 ~ 2'~

(2) INFORMATION FOR SEQ ID NO:32:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 34 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:

(2) INFORMATION FOR SEQ ID NO:33:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 3~ base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:34:

212~27 (2) INFORMATION FOR SEQ ID NO:34:
(i) SEQUENCE CHARACTERISTICS: .
(A) LENGTH: 34 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOCY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:34:
.. ..

(2) INFORMATION FOR SEQ ID NO:35:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32 base pairs (B) TYPE: nucleic acid -(C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:35:
' :

2 ~

(2) INFORMATION FOR SEQ ID NO:36:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 33 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:36:

(2) INFORMATION FOR SEQ ID NO:37:
(ii SEQUENCE CHARACTERISTICS:
(A) LENGTH: 34 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:37:

~ 74 ~
' 2 ~ ~J L,~ ~3 ~ ~

(2) INFORMATION FOR SEQ ID NO:38: ~ :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:38:

:
(2) INFORMATION FOR SEQ ID NO:39:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 33 base pairs (B) TYPE: nucleic acid .-(C) STRANDEDNESS: single (D) TOPOLOCY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:39:

212~3~i (2) INFORMATION FOR SEQ ID N0:40:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:40:

. (2) INFORMAT10N FOR SEQ ID N0:41:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 34 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID N0:41: ~-GGTACCGTCG ACTGAAAAGG TTGGCATCCA CCAT 34 ~ :

~:~2~02~

(2) INFORMATION FOR SEQ ID NO:42:
(i) SEQUENCE CHARACTERISTICS: ~ .
(A) LENGTH: 31 base pairs (B) TYPE: nucleic acid :: -(C) STRANDEDNESS: single :
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:42:

CAGCTCCGAT CCCCGTCTTT GAAGGCTATC T 31 ~.

(2) INF-ORMATION FOR SEQ ID NO:43:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 33 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear ::
(ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:43:

GGTACCGTCG ACACTCTGAT GGTACAGAAG CCT 33 -.

~ 1 2 ~

(2) INFORMATION FOR SEQ ID NO:44:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTN: 32 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:44:

(2) INFORMATION FOR SEQ ID NO:45: : :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 34 bass pairs ;~
(B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:45:

~ ~L 2 ~ J I , (2) INFORMAT!ON FOR SEQ ID NO:46:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 34 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:46:

CCCGGGGATC CGGCCCTCAT AACACCATGT GGGC 34 : ~ .
. .
: :
(2) INFORMATION FOR SEQ ID NO:47:
(i) SEQUENCE CHARACTERISTICS: :
(A) LENGTH: 32 base pairs (B) TYPE: n~cleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:47:

2~2~

(2) INFORMATION FOR SEQ ID NO:48:
(i) SEQUENCE CHARACTERISTICS:
(A) LENCTH: 32 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear ~:
(ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:48:

(2) INFORMATION FOR SEQ ID NO:49:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 28 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi~ SEQUENCE DESCRIPTION: SEQ ID NO:49: ;

Claims (11)

1. A polypeptide comprising at least 6 consecutive amino acids in the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID
NO: 3.

2. The polypeptide as claimed in claim 1, which is a non-A non-B hepatitis virus-constituting polypeptide represented by SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3.

3. The polypeptide as claimed in claim 1, which is represented by SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID
NO: 9.

4. DNA comprising at least 10 consecutive bases in the base sequence represented by SEQ ID NO: 4, SEQ
ID NO: 5 or SEQ ID NO: 6.

5. The DNA as claimed in claim 4, which encodes the polypeptide represented by SEQ ID NO: 1, SEQ ID
NO: 2 or SEQ ID NO: 3. and is represented by SEQ ID
NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6.

6. An immunochemical reagent for detecting anti non-A non-B hepatitis virus antibody in a biological sample which contains the polypeptide(s) as claimed in claim 1.

7. The immunochemical reagent for detecting anti non-A non-B hepatitis virus antibody as claimed in claim 6, which contains one or more polypeptide(s) selected from the group consisting of polypeptides represented by SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID
NO: 9.

8. A reagent for detecting non-A non-B
hepatitis virus gene which contains the DNA as claimed in claim 4.

9. A polyclonal antibody against the polypeptide as claimed in claim 1 as an antigen.

10. A monoclonal antibody against the polypeptide as claimed in claim 1 as an antigen.

11. A non-A non-B hepatitis virus vaccine produced by using the polypeptide as claimed in claim 1.